A mammalian liver contains enzymes that convert various chemical compositions to products which can be more easily eliminated from the body. This conversion process, which is also known as chemical metabolism or chemical biotransformation, frequently determines the duration of action of pharmaceuticals or the intensity of the pharmaceutical action. For this reason, many pharmaceuticals must typically be taken several times each day to treat diseases and produce other desirable pharmacological effects.
The liver includes many pharmaceutical-metabolizing enzyme systems such as cytochrome P450, carboxylesterases, UDP-glucuronosyltransferases, sulfotransferases, glutathione S-transferases and many others. These enzyme systems contain numerous individual enzymes, each of which is capable of metabolizing a wide variety of pharmaceuticals and other chemical compositions. Of these various enzyme systems, the P450 enzymes play a major role in determining the rate of elimination of drugs.
Cytochrome P450 enzymes comprise a large family of proteins (Nelson et al 1996) generally called isozymes or isoenzymes. Other new members of this family are still being discovered. In fact, relatively few species have been examined to this date, but, it is expected that the total number of enzymes will eventually be determined to be quite large. Current molecular biological techniques allow the generation of mutants of existing P450 enzymes and the construction of fusion proteins containing parts of P450 enzymes. These generated or constructed proteins still retain P450 function and as such are considered P450 proteins herein. Although liver cells (hepatocytes) contain the most cytochrome P450, other extrahepatic tissues also express other important isozymes, for example adrenals. The primary site for cytochrome P450 isozymes in hepatocytes is the endoplasmic reticulum.
Cytochrome P450 catalytic activity is considered a biotransformation reaction because substrates are changed in some way by the action of the enzyme. Three examples include introduction of a hydroxyl or epoxide functional group and dealkykation. In most cases of P450 biotransformation a source of electrons and molecular oxygen is required. Although hydroperoxides can substitute for both oxygen and electrons, in in vitro systems.
Without cytochrome P450 and related enzymes, naturally occurring and man-made foreign chemicals would accumulate in the body. However, biological or toxic effects of some chemicals are due to metabolites generated by cytochrome P450 and/or related enzymes. For example, the pharmacological effects of the anti-histamine, Seldane, are not due to its main ingredient, terfenadine, but are instead due to a metabolite of terfenadine that is generated by cytochrome P450. Similarly, the liver toxicity that can result from taking acetaminophen, the active ingredient in tylenol, is not due to acetaminophen per se, but is due to a toxic metabolite that is generated by cytochrome P450.
Metabolism by cytochrome P450 often represents the rate-limiting step in pharmaceutical elimination. Consequently, factors that lessen the activity of P450 enzymes usually prolong the effects of pharmaceuticals, whereas factors that increase cytochrome P450 activity have the opposite effect.
Changes in pharmaceutical metabolism may have undesirable or toxic consequences. For example, impaired metabolism of a pharmaceutical by factors that decrease cytochrome P450 activity may lead to symptoms of pharmaceutical overdose. In particular, the anti-coagulant warfarin can cause bleeding disorders when administered to individuals with low cytochrome P450 activity. Since the ulcer treatment drug cimetidine depresses P450 activity, warfarin is not administered to patients on cimetidine. Conversely, the accelerated metabolism of a drug due to increased concentrations of cytochrome P450 can also lead to a lessening of therapeutic effect. For example, pharmaceuticals such as phenobarbital and rifampin that increase cytochrome P450 activity lead to an increased rate of metabolism of contraceptive steroids. When the contraceptive steroids are consumed ovulation, and pregnancy, can result.
The liver converts many chemicals other than pharmaceuticals to metabolites that can be more readily eliminated from the body. Cytochrome P450 and related enzymes facilitate the elimination of endobiotics and of foreign chemicals called xenobiotics. To perform this function in their native environments, other components are often required such as other proteins, lipids, oxygen and electrons. Xenobiotics include environmental pollutants, pesticides, industrial chemicals, household products, cosmetics and non-nutrients in food such as plant alkaloids, flavorings, and chemicals that form during spoilage or cooking. Endobiotics are chemicals made in the body, such as steroid hormones, ecosanoids, and fat-soluble vitamins.
Cytochrome P450 biotransformation of different substrates including endogenous molecules and drugs can have a profound effect on the pharmacological and toxicological responses observed in different species. Because of this fact, pharmaceutical companies spend a considerable amount of their research time identifying the role of drug metabolizing enzymes, in particular cytochrome P450 enzymes, in the metabolism and disposition of novel therapeutics.
The need to determine if an enzyme or enzymes metabolize a drug is long standing. Information on P450 enzymes metabolizing a drug can be used to predict a variety of drug reactions. When administered with ketoconazole or erythromycin, the anti-histamine Seldane (active ingredient, terfenadine) causes Torsades de Points, which in some individuals leads to ventricular arrhythmias and heart failure. Terfenadine is extensively metabolized by intestinal and hepatic enzymes. When enzymes are inhibited by ketoconazole or erythromycin, the plasma levels of terfenadine become sufficiently elevated to block cardiac potassium channels. Such blockage may cause fatal ventricular arrhythmias.
Different sources have been used to measure P450 biotransformation reactions such as whole animals, tissue samples, cell fractions, and highly purified P450s. For example:
i) Radiolabelled drugs such as erythromycin and caffeine can be administered to human patients and P450 biotransformation capacity estimated from the amount of radiolabelled CO.sub.2 exhaled. Drugs are administered to humans and biotransformed by P450 enzymes and the rate of biotransformation determined by measuring parent drug or metabolite concentrations in urine or plasma samples. PA1 ii) Some techniques require the use of whole organ perfusions, tissue slices and cultured cells. PA1 iii) Microsomes, a crude biochemical preparation in which membrane fragments of the endoplasmic reticulum have been selectively enriched from other components of the cell, are prepared from a variety of tissues and are then used as a common source of material. More unrefined techniques use crude cell fractions such as a "S9" fraction. PA1 iv) Recombinant P450s may be generated in a variety of different heterologous expression systems, e.g. insect cells, yeast, E. coli. These eukaryotic and bacterial cells are used to over-express P450s and drug metabolizing enzymes. The cells are then used in a similar fashion as described in ii-iii. PA1 v) Highly purified P450 isozymes have been isolated from both human tissue samples and from heterologous expression systems, however, the P450 does not perform biotransformation reactions alone. The functional biotransformation activity of the P450 has to be reconstituted. In the simplest reconstitution scenario a P450 isozyme is mixed with a substrate and a hydroperoxide chemical (which serves as an electron donor) only then can biotransformation occur. PA1 i) it overcomes the perceived difficulties associated with performing P450 reconstitution reactions; PA1 ii) the error in biotransformation activity is lowered due to a significant decrease in pipetting manipulations; PA1 iii) the ratio of the individual purified enzyme components can easily be optimized to suit specific experimental requirements; PA1 iv) the results demonstrate the feasibility of making similar compositions for other P450 enzymes; PA1 v) a system is provided where catalysis by other drug metabolizing enzymes is prevented allowing for the rapid identification of novel P450 substrates and inhibitors and the development of large scale screening assays.
Traditionally, reconstitution of P450 biotransformation activity has been more complex and has involved the addition of several other components. For example a typical reconstitution reaction might consist of a purified P450, another protein termed P450 reductase (which supplies electrons to the P450) a lipid component, a substrate and an electron source such as NADPH.
Another example indicates how complex some biotransformation reconstitution reactions can be. Using a purified cytochrome P450 which possesses optimal activity toward a substrate testosterone named CYP3A4, three highly purified lipids were pretreated to form a liposomes complex. The liposome complex was then mixed with the purified P450, P450 reductase and another protein cytochrome b.sub.5. Other components at predetermined concentrations such as CHAPS, MgCl.sub.2, GSH, were also required for optimal biotransformation capacity. Most importantly, the way in which all the components were combined was critical for obtaining useable biotransformation capacity. For example, mixing the protein and lipids was the first step and other components for optimal catalytic activity were added at specific points in time. Multiple variations of this reconstitution technique exist in the literature including different ratios of catalytic enzymes, P450 enzymes, lipids, detergent, buffer components, sonicated lipids, nonsonicated lipids, separation of vesicles containing proteins from unincorporated proteins and proteins mixed at high concentrations in the absence of lipid.
Accordingly, it is desirable to provide a method for simplifying the identification of particular drugs and chemicals which are metabolized by a cytochrome P450 enzyme for use in preventing or modifying the administration of the drug to individuals having abnormal concentrations of the enzymes which metabolize the drug.